Rapamycin (sirolimus) (FIG. 1) is a lipophilic macrolide produced by Streptomyces hygroscopicus NRRL 5491 (Sehgal et al., 1975; Vézina et al., 1975; U.S. Pat. Nos. 3,929,992; 3,993,749) with a 1,2,3-tricarbonyl moiety linked to a pipecolic acid lactone (Paiva et al., 1991). Other related macrolides (FIG. 2) include FK506 (tacrolimus) (Schreiber and Crabtree, 1992), FK520 (ascomycin or immunomycin) (Wu et al., 2000), FK525 (Hatanaka H, et al., 1989), FK523 (Hatanaka, H., et al., 1988), antascomicins (Fehr, T., et al., 1996) and meridamycin (Salituro et al., 1995). For the purpose of this invention rapamycin is described by the numbering convention of McAlpine et al. (1991) in preference to the numbering conventions of Findlay et al. (1980) or Chemical Abstracts (11th Cumulative Index, 1982-1986 p 60719CS).
The polyketide backbone of rapamycin is synthesised by head-to-tail condensation of a total of seven propionate and seven acetate units to a shikimate derived cyclohexanecarboxylic acid starter unit (Paiva et al., 1991). The L-lysine derived amino acid, pipecolic acid, is condensed via an amide linkage onto the last acetate of the polyketide backbone (Paiva et al., 1993) and is followed by lactonisation to form the macrocycle. A 107 kb genomic region containing the biosynthetic gene cluster has been sequenced (Schwecke et al., 1995). Analysis of the open reading frames revealed three large genes encoding the modular polyketide synthase (PKS) (Aparicio et al., 1996; Schwecke et al., 1995). Embedded between the PKS genes lies the rapP gene encoding a protein with sequence similarity to activation domains of nonribosomal peptide synthetases and it is thought to act analogously (König et al., 1997). The region encoding the PKS genes is flanked on both sides by 24 additional open reading frames encoding enzymes believed to be required for the biosynthesis of rapamycin (Molnár et al., 1996). These include the following post-polyketide modification enzymes: two cytochrome P-450 monooxygenases, designated as RapJ and RapN, an associated ferredoxin RapO, and three SAM-dependent O-methyltransferases RapI, RapM and RapQ. Other adjacent genes have putative roles in the regulation and the export of rapamycin (Molnár et al., 1996). The cluster also contains the gene rapL whose product RapL is proposed to catalyse the formation of the rapamycin precursor L-pipecolic acid through the cyclodeamination of L-lysine (Khaw et al., 1998; Paiva et al., 1993).
The polyketide core of rapamycin is assembled by the very large, multifunctional proteins that comprise the Type I polyketide synthase (rap PKS). This polypeptide complex comprises a loading module and fourteen extension modules, each module being responsible for both the addition of a specific acyl-CoA precursor to the growing polyketide chain, and for the degree of reduction of the β-keto carbonyl group. Each module performs several biochemical reactions which are carried out by specific domains of the polypeptide. All the extension modules contain an acyl transferase (AT) domain which selects and then donates the acyl group from a precursor to an acyl carrier protein (ACP) domain, and a β-ketosynthase (KS) domain that adds the pre-existing polyketide chain to the new acyl-ACP by decarboxylative condensation. Additional domains are present in some extension modules: β-ketoreductase (KR) domains which reduce β-keto groups to hydroxyls, dehydratase (DH) domains which act on hydroxyls to leave double bonds, and enoyl reductase (ER) domains which reduce double bonds to leave saturated carbons. In modules 3 and 6 the β-keto processing domains that are present are predicted to be inactive, as the action of these domains is not reflected in the ultimate structure. The final extension module (extension module 14) appears not to contain any β-keto-processing domains. The initiation of rapamycin biosynthesis occurs via incorporation of 4,5-dihydroxycyclohex-1-enecarboxylic acid that is derived from the shikimate pathway (Lowden, P. A. S., et al. 2001) and is common to other FKBP-binding molecules such as FK506 and FK520 (FIG. 2). Following biosynthesis of the polyketide, the NRPS module encoded by rapP, incorporates pipecolic acid (L-lysine derived), which is condensed via an amide linkage onto the last acetate of the polyketide backbone (Paiva et al., 1993). This is followed by lactonisation to form the macrocycle.
The nucleotide sequences for each of the three rapamycin PKS genes, the NRPS-encoding gene and the flanking late gene sequences and the corresponding polypeptides, are identified in Aparicio et al., 1996, and Schwecke et al., 1995 and are deposited with the NCBI under accession number X86780, and corrections to this sequence have recently been published in WO 04/007709.
The first enzyme-free product of the rapamycin biosynthetic cluster has been designated pre-rapamycin (WO 04/007709, Gregory et al., 2004). Production of the fully processed rapamycin requires additional processing of the polyketide/NRPS core by the enzymes encoded by the rapamycin late genes, RapJ, RapN, RapO, RapM, RapQ and RapI.
Rapamycin has significant pharmacological value due to the wide spectrum of activities exhibited by the compound; this emphasizes the necessity to generate novel analogues of the drug. Rapamycin shows moderate antifungal activity, mainly against Candida species but also against filamentous fungi (Baker et al., 1978; Sehgal et al., 1975; Vézina et al., 1975; U.S. Pat. Nos. 3,929,992; 3,993,749). Rapamycin inhibits cell proliferation by targeting signal transduction pathways in a variety of cell types, e.g. by inhibiting signalling pathways that allow progression from the G1 to the S-phase of the cell cycle (Kuo et al., 1992). In T cells rapamycin inhibits signalling from the IL-2 receptor and subsequent autoproliferation of the T cells resulting in immunosuppression. The inhibitory effects of rapamycin are not limited to T cells, since rapamycin inhibits the proliferation of many mammalian cell types (Brunn et al., 1996). Rapamycin is, therefore, a potent immunosuppressant with established or predicted therapeutic applications in the prevention of organ allograft rejection and in the treatment of autoimmune diseases (Kahan et al., 1991). 40-O-(2-hydroxy)ethyl-rapamycin (SDZ RAD, Certican, everolimus) is a semi-synthetic analogue of rapamycin that shows immunosuppressive pharmacological effects (Sedrani, R. et al., 1998; Kirchner et al., 2000; U.S. Pat. No. 5,665,772). Approval for this drug was obtained for Europe in 2003 and it is expected to be launched in the US shortly. The rapamycin ester CCI-779 (Wyeth-Ayerst) inhibits cell growth in vitro and inhibits tumour growth in vivo (Yu et al., 2001). CCI-779 is currently in Phase III clinical trials. The value of rapamycin in the treatment of chronic plaque psoriasis (Kirby and Griffiths, 2001), the potential use of effects such as the stimulation of neurite outgrowth in PC12 cells (Lyons et al., 1994), the block of the proliferative responses to cytokines by vascular and smooth muscle cells after mechanical injury (Gregory et al., 1993) and its role in prevention of allograft fibrosis (Waller and Nicholson, 2001) are areas of intense research (Kahan and Camardo, 2001). Recent reports reveal that rapamycin is associated with a lower incidence of cancer in organ allograft patients on long-term immunosuppressive therapy than those on other immunosuppressive regimes, and that this reduced cancer incidence is due to inhibition of angiogenesis (Guba et al., 2002). It has been reported that the neurotrophic activities of immunophilin ligands are independent of their immunosuppressive activity (Steiner et al., 1997) and that nerve growth stimulation is promoted by disruption of the mature steroid receptor complex as outlined in the patent application WO 01/03692. Side effects such as hyperlipidemia and thrombocytopenia as well as potential teratogenic effects have been reported (Hentges et al., 2001; Kahan and Camardo, 2001).
Rapamycin impacts signalling cascades within the cell through the inhibition of the p70S6k kinase, a serine/threonine kinase in higher eukaryotes that phosphorylates the ribosomal protein S6 (Ferrari et al., 1993; Kuo et al., 1992). The S6 protein is located in the ribosomal 40S subunit and it is believed to be an important functional site involved in tRNA and mRNA binding. A regulatory function for mRNA translation through S6 phosphorylation by p70S6k has been postulated (Kawasome et al., 1998). Rapamycin inhibits protein synthesis through its effect on other growth related events, including the activity of cyclin-dependent kinases, phosphorylation of cAMP-responsive element modulator (CREM) and phosphorylation of the elongation factor binding protein 4E-BP1 (PHAS1) (Hung et al., 1996). The drug induces the accumulation of the dephosphorylated species of 4E-BP1 that binds to the translation initiation factor eIF-4E, thus, suppressing translation initiation of cap-dependent mRNAs (Hara et al., 1997; Raught et al., 2001).
The pharmacologic actions of rapamycin characterised to date are believed to be mediated by the interaction with cytosolic receptors termed FKBPs or immunophilins. Immunophilins (this term is used to denote immunosuppressant binding proteins) catalyse the isomerisation of cis and trans peptidyl-proline bonds and belong to a highly conserved family of enzymes found in a wide variety of organisms (Rosen and Schreiber, 1992). Two large groups of enzymes belonging to the family of immunophilins are represented by FKBPs and cyclophilins (Schreiber and Crabtree, 1992). The major intracellular rapamycin receptor in eukaryotic T-cells is FKBP12 (DiLella and Craig, 1991) and the resulting complex interacts specifically with target proteins to inhibit the signal transduction cascade of the cell. Analysis of the crystal structure of the FKBP12-rapamycin complex has identified a rapamycin-binding pharmacophore termed the ‘binding domain’ (Van Duyne et al., 1993) (see FIG. 1). The ‘binding domain’ is required for the interaction with the immunophilin and consists of the C-1 to C-14 region including the ester linkage, the pipecolic acid-derived ring, the dicarbonyl and the hemiketal ring. The interaction is characterised by many hydrophobic contacts and some hydrogen bonds including one to the hydroxyl group on the cyclohexane ring. The pipecolinyl ring (C2 to N7) makes the deepest penetration into the protein where it is surrounded by highly conserved aromatic amino acid residues lining the hydrophobic binding cavity. Both the C1 and the C8 carbonyl groups are involved in hydrogen bonding and the C9 carbonyl group protrudes into a pocket formed by three completely conserved aromatic amino acid residues (one tyrosine and two phenylalanine acid residues) in FKBP12. The domain of the immunophilin-ligand complex which interacts with the target proteins projects away from FKBP.
Most immunophilins do not appear to be directly involved in immunosuppressive activities and relatively little is known concerning their natural ligands although candidates for natural ligands of the FKBPs termed FKBP-associated proteins (FAP) such as FAP48 and FAP1 have been reported. The specific interaction of FAPs with FKBPs during the formation of complexes was prevented by rapamycin in a dose-dependent manner (Chambraud et al., 1996; Kunz et al., 2000). Immunophilins appear to function in a wide range of cellular activities such as protein folding; assembly and trafficking of proteins; co-regulation of molecular complexes including heat shock proteins; steroid receptors; ion channels; cell-to-cell interactions and transcription and translation of genes (Galat 2000; Hamilton and Steiner 1998). All immunophilins possess the protein folding property of peptidyl-prolyl cis-trans isomerisation and several immunophilins are found located in the endoplasmic reticulum, a principal site of protein synthesis in the cell. In addition to FKBP12 (U.S. Pat. No. 5,109,112) other immunophilins include FKBP12.6 (U.S. Pat. No. 5,457,182), FKBP13 (Hendrickson et al., 1993; U.S. Pat. No. 5,498,597), FKBP25 (Hung and Schreiber, 1992; Jin et al., 1992), FKBP14.6 (U.S. Pat. No. 5,354,845), FKBP52 (U.S. Pat. No. 5,763,590), FKBP60 (Yem et al., 1992) and FKBP65 (Patterson et al., 2000).
The target of the rapamycin-FKBP12 complex has been identified in yeast as TOR (target of rapamycin) (Alarcon et al., 1999) and the mammalian protein is known as FRAP (FKBP-rapamycin associated protein) or mTOR (mammalian target of rapamycin) (Brown et al., 1994). These proteins show significant similarity to the phosphotransferase domains of phosphatidylinositol 3-kinases and the observation that a point mutation in the FKBP12-rapamycin binding domain (FRB) of mTOR abolishes mTOR kinase activity provides evidence for the involvement of FRB in the function of the kinase domain (Vilella-Bach et al., 1999). The crystal structure of FKBP12-rapamycin with a truncated form of mTOR containing the FRB domain (Chen et al., 1995) has been obtained thus defining the ‘effector’ domain of rapamycin (Choi et al., 1996; Liang et al., 1999). The analysis of the crystal structure revealed that protein-protein contacts are relatively limited compared to the interaction between rapamycin and each protein. No hydrogen bonds between rapamycin and FRB were identified. Interaction is concentrated in a series of hydrophobic contacts between the triene region of rapamycin and mainly aromatic residues of FRB (Liang et al., 1999). The most deeply buried atom of rapamycin is the methyl attached to C23 (see FIG. 1). The C23 to C34 region and the cyclohexyl ring of rapamycin make hydrophobic contacts with FRB. A small conformational change in rapamycin was evident between the binary and the ternary complexes (Liang et al., 1999).
Divergences between the biological effects of rapamycin analogues modified at the C16 methoxy group and their ability to bind FKBP12 were detected and the location of the C16 substituents at the interfacial space between FKBP12 and mTOR was postulated (Luengo et al., 1995). The analysis of the crystal structure of FKBP12 with the non-immunosuppressive 28-O-methyl rapamycin revealed a significant difference in the orientation of the cyclohexyl ring which may result in disruption of mTOR binding (Kallen et al., 1996).
A link between mTOR signalling and localized protein synthesis in neurons; its effect on the phosphorylation state of proteins involved in translational control; the abundance of components of the translation machinery at the transcriptional and translational levels; control of amino acid permease activity and the coordination of the transcription of many enzymes involved in metabolic pathways have been described (Raught et al., 2001). Rapamycin sensitive signalling pathways also appear to play an important role in embryonic brain development, learning and memory formation (Tang et al., 2002). Research on TOR proteins in yeast also revealed their roles in modulating nutrient-sensitive signalling pathways (Hardwick et al., 1999). Similarly, mTOR has been identified as a direct target for the action of protein kinase B (akt) and of having a key role in insulin signalling (Shepherd et al., 1998; Navé et al., 1999). Mammalian TOR has also been implicated in the polarization of the actin cytoskeleton and the regulation of translational initiation (Alarcon et al., 1999). Phosphatidylinositol 3-kinases, such as mTOR, are functional in several aspects of the pathogenesis of tumours such as cell-cycle progression, adhesion, cell survival and angiogenesis (Roymans and Slegers, 2001).
The multitude of the FKBP's which are present in different cell types also underline the utility of isolating novel FKBP-ligand analogues with potentially changed binding and/or effector domains. For example, it is the inhibition of the rotamase enzyme FKBP52, which forms part of the steroid receptor complex, which has been identified as the mechanism by which rapamycin analogues (and other FKBP12-binding compounds) moderate neural regeneration and neurite outgrowth (WO 01/03692)
Pharmacokinetic studies of rapamycin and rapamycin analogues have demonstrated the need for the development of novel rapamycin compounds that may be more stable in solution, more resistant to metabolic attack and/or have improved bio-availability. Modification of rapamycin using the chemically available positions has been extensively addressed (see below). However this approach is restricted to the few sites available for chemical modification and is further limited in its utility by difficulties in selective modification at a particular position in the presence of other reactive sites on the molecule.
A range of synthesised rapamycin analogues using the chemically available sites of the molecule has been reported. The description of the following compounds was adapted to the numbering system of the rapamycin molecule described in FIG. 1. Chemically available sites on the molecule for derivatisation or replacement include C40 and C28 hydroxyl groups (e.g. U.S. Pat. Nos. 5,665,772; 5,362,718), C39 and C16 methoxy groups (e.g. WO 96/41807; U.S. Pat. No. 5,728,710), C32, C26 and C9 keto groups (e.g. U.S. Pat. Nos. 5,378,836; 5,138,051; 5,665,772). Hydrogenation at C17, C19 and/or C21, targeting the triene, resulted in retention of antifungal activity but relative loss of immunosuppression (e.g. U.S. Pat. Nos. 5,391,730; 5,023,262). Significant improvements in the stability of the molecule (e.g. formation of oximes at C32, C40 and/or C28, U.S. Pat. Nos. 5,563,145, 5,446,048), resistance to metabolic attack (e.g. U.S. Pat. No. 5,912,253), bioavailability (e.g. U.S. Pat. Nos. 5,221,670; 5,955,457; WO 98/04279) and the production of prodrugs (e.g. U.S. Pat. Nos. 6,015,815; 5,432,183) have been achieved through derivatisation. However, chemical modification requires significant quantities of rapamycin template and, as a base and acid labile compound, it is difficult to work with. While chemical derivatisation can be group selective, it can often be difficult to be site selective. Consequently, chemical modification often requires multiple protective and deprotective steps and can produce mixed products in variable yields.
Biological approaches to producing novel rapamycin analogues were initially slow to be productive due to the difficulties encountered in working with the producing organism (Lomovskaya et al., 1997; Kieser et al., 2000) despite the availability of the sequence of the biosynthetic gene cluster of rapamycin from S. hygroscopicus (Aparicio et al., 1996; Schwecke et al., 1995). A recent patent application from the present inventors describes a wider range of rapamycin analogues than had been previously accessible via modification of the rapamycin biosynthetic pathway by manipulation of the post PKS modifying genes (WO 04/007709). Further analogues can also be accessed by feeding alternatives to the natural starter acid of the rapamycin PKS which are incorporated into the rapamycin structures (WO 04/007709). While these methods provide access to significantly more of the chemical space around the rapamycin molecule, this technology does not allow the modification of the core framework of the rapamycin molecule that is encoded by the type I polyketide synthase genes.
The isolation of rapamycin analogues using other biological methods such as biotransformation and phage-based genetic modification has also been described. Isolation of minor metabolites from both mutant strains and rapamycin producing strains has provided small quantities of a number of rapamycin analogues. These strains are often low yielding and produce mixtures of rapamycin analogues. The isolation of 27-O-desmethylrapamycin and 27-desmethoxyrapamycin was reported from the culture supernatant of S. hygroscopicus NCIMB 40319 (Box et al., 1995). The antifungal activity of 27-O-desmethylrapamycin was lower than that of rapamycin but the inhibition of FKBP12 PPlase activity seemed to be increased. The inhibition of ConA-stimulated proliferation of murine splenic T cells and the inhibition of LPS-stimulated proliferation of murine splenic B cells was decreased when compared to rapamycin (Box et al., 1995). Similarly, antifungal activities of the rapamycin analogues prolylrapamycin, 27-O-desmethylrapamycin and 27-desmethoxyrapamycin (numbering system of the rapamycin molecule as described in FIG. 1) were lower than that of rapamycin (Wong et al., 1998). Rapamycin analogues (16-O-desmethylrapamycin, 27-O-desmethylrapamycin, 39-O-desmethylrapamycin, 16,27-O-bisdesmethylrapamycin, prolylrapamycin, 26-O-desmethylprolylrapamycin, 9-deoxorapamycin, 27-desmethoxyrapamycin, 27-desmethoxy-39-O-desmethylrapamycin, 9-deoxo-27-desmethoxyrapamycin, 28-dehydrorapamycin, 9-deoxo-27-desmethoxy-39-O-desmethylrapamycin) were also isolated from Actinoplanes sp N902-109 after the addition of cytochrome P450 inhibitors and/or precursor feeding to the culture or after biotransformation of isolated rapamycin (Nishida et al., 1995). The use of such inhibitors, however, only allows the targeting of a particular enzyme function and is not site selective thus often resulting in mixtures of products. Rational production of a single selected analogue is not possible via this method. The resulting production of mixtures of rapamycin analogues rather than a single desired product also impacts yield. The mixed lymphocyte reaction (MLR) inhibitory activity of the compounds was assessed and little effect on the activity was detected after the loss of the methyl group at C27 or/and C16. A more significant decrease in activity was observed for 9-deoxorapamycin; additionally, the loss of the methoxy group at C27, the hydroxy group at C28 and the substitution of a pipecolic acid-derived group with a prolyl group all resulted in a reduction in potency (Nishida et al., 1995). Similarly, biotransformation of rapamycin and the isolation of 16,39-O-bisdesmethylrapamycin have been reported (WO 94/09010). The retention of some inhibitory activity in cell proliferation assays with compounds modified in the cyclohexyl ring, e.g. 39-O-desmethylrapamycin and C40 modifications such as SDZ RAD and CCI-779, identify this region of the molecule as a target for the generation of novel rapamycin analogues both with immunosuppressive and anti-cancer properties.
Novel rapamycin analogues have been reported after feeding cyclohexanecarboxylic acid, cycloheptanecarboxylic acid, cyclohex-1-enecarboxylic acid, 3-methylcyclohexanecarboxylic acid, cyclohex-3-enecarboxylic acid, 3-hydroxycyclohex-4-enecarboxylic acid and cyclohept-1-enecarboxylic acid to cultures of rapamycin-producing wild type S. hygroscopicus thus demonstrating the flexibility in the loading module of the rapamycin polyketide synthase (P. A. S. Lowden, Ph.D dissertation, University of Cambridge, 1997; Lowden et al., 2004). These novel rapamycin analogues were produced in competition with the natural starter, 4,5-dihydroxycyclohex-1-enecarboxylic acid, resulting in reduced yields and mixed products.
Two novel sulphur-containing rapamycin analogues of rapamycin have been isolated by feeding cultures of the rapamycin-producing S. hygroscopicus NRRL5491 with (±) nipecotic acid, to inhibit L-pipecolic acid production, and co-feeding the sulphur-containing pipecolate analogues (S)-1,4-thiazane-3 carboxylic acid or (S)-1,4-thiazane-4 carboxylic acid (Graziani et al., 2003).
The isolation of two recombinant S. hygroscopicus strains producing various rapamycin analogues, using biological methods mediated by phage technology (Lomovskaya et al., 1997), has been reported. In the presence of added proline analogues, a S. hygroscopicus rapL deletion mutant synthesized the novel rapamycin analogues prolylrapamycin, 4-hydroxyprolylrapamycin and 4-hydroxyprolyl-26-desmethoxy-rapamycin (Khaw et al., 1998). Similarly, the novel rapamycins 3-hydroxy-prolyl-rapamycin, 3-hydroxy-prolyl-26-desmethoxy-rapamycin, and trans-3-aza-bicyclo[3,1,0]hexane-2-carboxylic acid rapamycin have been identified from fed cultures of the same mutant strain as described in WO 98/54308. The activity of prolylrapamycin and 4-hydroxyprolyl-26-desmethoxy-rapamycin was assessed in proliferation assays and the inhibitory activity of the latter compound was significantly less than that of rapamycin (Khaw et al., 1998). The deletion of the five contiguous genes, rapQONML (responsible for post-polyketide modifications at C16, C27 and production of L-pipecolic acid) and their replacement with a neomycin resistance marker in S. hygroscopicus ATCC29253 using phage-based methodology resulted in the production of 16-O-desmethyl-27-desmethoxyrapamycin when fed with pipecolic acid (Chung et al., 2001). No complementation of this deletion mutant has been demonstrated. Furthermore, the site-specificity of neither RapM nor RapQ was identified in this work; therefore, rational design of rapamycin analogues requiring methylation at C16-OH or C27-OH was not enabled by Chung et al. (2001). The phage-based methodology suffers from a number of drawbacks, as described in more detail below, exemplified by the reported production of only three recombinant S. hygroscopicus strains over a period of 9 years from the disclosure of the gene sequence. It offers a difficult and protracted process of obtaining engineered strains and is significantly more limited in utility in comparison to the methodology disclosed within the earlier application by the present inventors (WO 04/007709).
Conventional approaches to manipulate rapamycin modifying genes using biological methods comprise the mutation or deletion of individual genes in the chromosome of a host strain or/and the insertion of individual genes as extra copies of homologous or heterologous genes either individually or as gene cassettes (WO 01/79520, WO 03/048375). However, the isolation of novel rapamycin analogues using such biological methods has been limited due to the difficulties in transforming the rapamycin-producing organism S. hygroscopicus. It has been reported that the commonly used methods of transformation with plasmid DNA or conjugal transfer were unsuccessful with the rapamycin producing strain (Lomovskya et al., 1997, Schwecke et al., 1995, Kieser et al., 2000). The state of the art prior to the disclosure of WO 04/007709 used the methodology of Lomovskya et al. (1997), a work intensive phage based method that is severely limited by the size of the cloned DNA fragments transferred into S. hygroscopicus (Kieser et al., 2000). This technology is limited to the transfer of a maximum of approximately 6.4 kbp of cloned DNA. Thus, when complementing a deletion mutant using this technology the artisan is limited to the inclusion of genetic material within this size limit, for example complementation is limited to two typical functional genes (usually approximately 1 kbp each in size) in addition to a desired promoter, regions of homology (if required) and resistance marker. WO 04/007709 disclosed the first description of recombinant technology methods for the efficient transformation of strain such as Streptomyces hygroscopicus subsp. hygroscopicus NRRL5491 that contains a rapamycin biosynthetic gene cluster. Prior to this, although the genetic information for the rapamycin biosynthetic gene cluster has been available since 1995 (Schwecke et al., 1995), limited progress in this area had hitherto been made (Khaw et al., 1998; Chung et al., 2001; WO 01/34816). WO 04/007709 describes methods for manipulating the rapamycin biosynthetic pathway and producing a number of rapamycin analogues by complementation of a deletion mutant in which the amount of post-PKS processing is specifically varied, and optionally feeding exogenous starter acids to strains in which at least rapK has been deleted or inactivated.
Previous work has demonstrated that polyketide synthase (PKS) genes can, in principle, be manipulated with the objective of providing novel polyketides. These alterations include:                Deletions: In-frame deletion of the DNA encoding part of the KR domain in module 5 of the erythromycin-producing (ery) PKS in Saccharopolyspora erythraea has been shown to lead to the formation of erythromycin analogues, namely 5,6-dideoxy-3-α-mycarosyl-5-oxoerythronolide B and 5,6-dideoxy-5-oxoerythronolide B (Donadio et al., 1991).        Inactivation of individual domains: alteration of active site residues in the ER domain of module 4 of the ery PKS, by genetic engineering of the corresponding PKS-encoding DNA and its introduction into Saccharopolyspora erythraea, led to the production of 6,7-anhydroerythromycin C (Donadio et al., 1993).        Loading module swaps: WO 98/01546 discloses replacement of the loading module of the ery PKS with the loading module from the avermectin (ave) PKS, to produce a hybrid Type I PKS gene that incorporates different starter units to make novel erythromycin analogues. A hybrid tylactone load/platenolide polyketide synthase was generated (Kuhstoss et al., 1996) which successfully transferred the specificity of the tylactone loading module for methylmalonyl-CoA to the platenolide molecule. The elucidation of the function of KSQ (Bisang et al., 1999) and manipulation of KSQ-containing loading modules is disclosed in WO 00/00618. Loading module swaps in the spinosyn biosynthetic pathway have also been described in WO 03/070908.        AT swaps: Oliynyk et al., (1996) and WO 98/01546 describe an AT domain swap where the erythromycin module 1 AT was replaced by the AT domain of rapamycin module 2 and the specificity of module 1 for the extender unit was altered accordingly. Further AT domain swaps have been described, for example in the erythromycin polyketide synthase (WO 98/01546, Ruan et al., 1997; Stassi et al., 1998) and in the spinosyn polyketide synthase (WO 03/070908).        Reductive Loop Swaps: for example alterations in the amount of reductive processing of the β-keto group formed during each condensation are described in WO 98/01546, WO 00/01827 and Kao et al., (1997).        Combinations of the domain swap approaches are exemplified in McDaniel et al., (1999).        Site-directed mutagenesis: WO 02/14482, Reeves et al., (2001), Reid et al., (2003) and Del Vecchio et al., (2003) demonstrate that it is possible to affect the substrate specificity of AT domains by selected mutation of specified residues.        Ring contraction: for example using an incomplete modular polyketide synthase (Kao et al. 1994) or moving the erythromycin chain terminating thioesterase domain downstream of modules 1, 2, 3, 5 or 6 (Cortés et al., 1995; Kao et al., 1995; Kao et al., 1996; Böhm et al., 1998) of the erythromycin PKS lead to the production of the expected truncated erythromycin molecules.        Ring expansion: insertion of a rapamycin module into the first ORF of the erythromycin PKS between erythromycin PKS modules 1 and 2 lead to production of the expected 16-membered macrolide (Rowe et al., 2001).        
Modifications of PKS clusters are not limited to those described above.
However, it has also been found that not all manipulations of PKS genes are capable of producing the predicted new analogues. When Donadio et al., (1993) inactivated an enoyl reductase (ER) domain of the erythromycin PKS, the resulting anhydro-analogue was not completely processed because it was no longer a substrate for the mycarose-O-methyltransferase. Similarly, changing the polyketide starter unit prevented complete elongation and elaboration of a rifamycin analogue in Amycolatopsis mediterranei (Hunziker et al., 1998).
If rapamycin analogues could be made by engineering the polyketide synthase genes in the biosynthetic cluster they would be highly desirable because they are predicted to have interesting biological activity.
The sequence of the rapamycin biosynthetic cluster was first published in 1995 (Schwecke et al.; Aparicio et al., 1996). Despite the wealth of prior art regarding the manipulation of PKSs described above there have to date been no reports of successful polyketide engineering of the core polyketide synthase of the rapamycin cluster. S. hygroscopicus, the rapamycin producer, is a difficult organism to manipulate (Lomovskaya et al., 1997).
A host cell which has been modified to produce 17-desmethylrapamycin is claimed in U.S. Pat. No. 6,670,168, however this patent lacks any description or working example of how this compound could be made, other than the hypothesis that such a strain could be constructed by the substitution of the AT domain of module 10. The fact that a substitution at this position could hypothetically lead to 17-desmethylrapamycin was obvious to any person of skill in the art, but it is not apparent how such a substitution could be made in light of the difficulties highlighted above, and no teaching to this effect was provided in U.S. Pat. No. 6,670,168. In light of the known difficulties of working with S. hygroscopicus and in the absence of any detailed description of how such a recombinant host strain could be generated it is clear that this is merely a description of what these authors hoped to achieve rather than being an enabling disclosure of such a strain. Additionally, U.S. Pat. No. 6,670,168 does not provide any teaching to a person of skill in the art regarding how 17-desmethylrapamycin could be prepared or isolated. The state of the art regarding the biosynthesis of rapamycin analogues is limited to WO 04/007709 in which the genes encoding post PKS modifying enzymes are manipulated and related applications which describe the feeding of exogenous starter acids for incorporation into rapamycin. The general lack of progress in engineering the PKS of rapamycin is due to the technological difficulties in transformation of the producing organism S. hygroscopicus NRRL5491.
The present invention is concerned with the generation of 17-desmethylrapamycin and analogues thereof. Accessing 17-desmethylrapamycin analogues requires the engineering of the rapamycin PKS. It is not obvious within the current state of the art that it is possible to achieve the engineering required to produce such a strain. In the present invention the initial target is the engineering of S. hygroscopicus MG2-10. S. hygroscopicus MG2-10 produces pre-rapamycin when fed with the exogenous acid 3,4-dihydroxycyclohexanecarboxylic acid as disclosed in WO 04/007709 and Gregory et al., (2004). Engineering of this organism was performed to generate an engineered strain which produces 17-desmethylpre-rapamycin when fed with the exogenous acid 3,4-dihydroxycyclohexanecarboxylic acid. The current state of the art teaches how such a strain may be used in further experiments as a base strain for complementation by late gene cassettes (WO 04/007709) and/or as a base strain for feeding exogenous acids (WO 04/007709) and generating further rapamycin analogues, but does not teach how such a strain may be generated in S. hygroscopicus by PKS engineering. The present invention describes, surprisingly, the successful application of PKS engineering methodologies, similar to those described in Oliynyk et al., 1996, to the rapamycin PKS. This is an unexpected result as these recombinant DNA technologies have not previously been successfully applied to S. hygroscopicus. The generation of a strain for production of 17-desmethylpre-rapamycin analogues is a useful base strain for complementation by late gene cassettes (WO 04/007709) and/or as a base strain for feeding exogenous acids (WO 04/007709). Each of these rapamycin analogues can then further be modified by semi-synthesis. This is the first demonstration of a truly combinatorial method which may be used to generate a library of rapamycin analogues may be altered at one or more of: the PKS engineering level, the auxiliary gene level, starter acid incorporation, amino acid incorporation and further at the accessible chemical functionality on the molecule, which remains accessible to semi-synthesis.